The present invention relates to analytical chemistry and, more particularly, to the preparation of gel columns such as those used in gel electrophoresis. A major objective of the present invention is the formation of a gel column which resists migration of the gel from its container and is essentially free of voids.
Much of recent progress in biotechnology, which holds great promise for advancing medicine and our understanding of life, is predicated on the ability to analyze the constituents of living organisms. In many cases it is necessary to separate constituents, e.g., proteins, to identify them and determine their relative concentrations in a sample.
Gel-column electrophoresis is one important separation methodology. During electrophoresis, an ionic sample is introduced at one end of a gel-filled column. The ionized components migrate longitudinally toward the other end of the column under the influence of an applied longitudinal electric field. The rate of migration for a ion is a function of its charge and bulk. Different chemical species are characterized by different bulks and charges so that they migrate at different rates. Since different species migrate at different rates, they can separate into distinct bands along the column.
The different species can be identified in several ways. In some cases, the final position within the column suffices to identify the chemical species. Similarly, species can be identified by the time at which bands elute from the column. In other cases, the chemical composition of the separated bands can be assessed by a technique such as spectrometry. Alternatively, the gel can be sliced and the bands subject to more intensive analysis.
Gel columns are typically formed by polymerizing a monomer solution in a fused silica tube. The inner wall of the tube is preferably pretreated with a bifunctional reagent. One functional group is capable of bonding to the wall, while the other remains available as a bonding site for the gel. Once the tube is so prepared, monomer solution is introduced and polymerization initiated. The resulting gel is securely bonded to the tube. This bonding resists migration of the gel from the tube during electrophoresis due to charges in the gel from impurities or partially hydrolyzed monomer functional groups. In addition, bonding minimizes the formation of non-sieving holes near the tube wall that can otherwise occur due to shrinkage that accompanies polymerization. However, shrinkage during polymerization can create voids in the form of air bubbles in the interior of the gel when the bifunctional reagent prevents the gel from separating from the tube wall. These voids disturb the electric fields and the migration of ions through the column, impairing separation.
Precompressing the monomer solution to a density about that of the completed gel prevents shrinkage during polymerization and thus the voids induced by the shrinkage, as taught by Bente and Myerson in U.S. Pat. No. 4,810,456. However, it can be difficult to work with the pressures, preferably around 8200 pounds per square inch, required for precompression. Furthermore, the resulting columns are subject to forming gel inhomogeneities or voids under application of moderate electric field strengths, e.g., 200 volts/centimeter, that can be used during microcapillary gel electrophoresis.
Another approach to forming void-free gel columns involves adding a hydrophillic polymer to the primary monomer solution, as disclosed by Karger et al. in U.S. Pat. No. 4,865,707. The rationale is that the resulting gel is more elastic and therefore more able to accommodate the stresses induced due to polymer shrinkage. However, the admixed hydrophillic polymer can adversely affect constituent separation during electrophoresis. Moreover, this approach has not reliably produced effective separation columns.
What is needed is an improved method and system for preparing electrophoretic gel columns. The method should not require the addition of foreign polymer components which adversely affect gel separation properties. Extreme pressures and other extreme ambient conditions should not be required. The resulting gel should be free of voids and should resist migration even when the higher voltages used for microcapillary electrophoresis are applied.